PI: Jan Dvorak, University of California, Davis Co-PI: Olin D. Anderson, UC Davis/USDA-ARS, Albany, CA Co-PI: Bikram S. Gill, Kansas State University Co-PI: Mingcheng Luo, University of California, Davis Senior Personnel: Wanlong Li, Kansas State University
Advances in wheat genetics and genomics are essential for the sustained development of wheat varieties with enhanced yield potential, resistance to pests and diseases, and tolerance of adverse environmental conditions. Wheat has an exceptionally large amount of DNA in its nucleus. Wheat is also polyploid; it originated by interspecific hybridization, and its nucleus contains three different genomes, designated A, B, and D, each contributed by a different diploid species.
The goal of this project is to develop genomic resources for physical mapping of wheat chromosomes and to deploy them in physical mapping of chromosomes of the wheat D genome. A physical map of a chromosome is a physical representation of the linear order of genes and other landmarks along the chromosome. To construct a physical map, genomic DNA is fragmented, DNA fragments are cloned and each clone is ?fingerprinted?. Overlaps between fingerprints are used to identify DNA fragments from neighboring regions of a chromosome. The location of genes and other markers in these fragments is determined and the sequence of overlapping DNA fragments is aligned to the linear order of markers along a chromosome. Physical maps are important for gene cloning, the development of genetic markers for tagging genes during breeding, and often a prerequisite for genome sequencing. Wheat polyploidy and large genomes require the use of novel strategies in the physical mapping of its chromosomes. Instead of attempting to construct wheat physical maps globally, the physical maps of the chromosomes of Aegilops tauschii, the diploid ancestor of the wheat D genome, will be constructed first. These maps will then be used as templates for the construction of the physical maps of three individual chromosomes of the wheat D genome in the cultivar Chinese Spring (CS). This step will be facilitated by international collaboration with the Institute of Experimental Botany (IEB), Czech Republic, which developed a technique for the isolation of individual wheat chromosomes by chromosome flow-sorting. This project complements ongoing national and international work toward wheat genome sequencing and it provides opportunities for training of US postdoctoral researchers and students at several educational levels.
Sequence-ready physical maps of the three CS chromosomes will make it theoretically possible to initiate wheat genome sequencing at the end of this project. While it will take other projects and an unknown number of years before the full wheat genomic sequence will be available to the research community, the anchored physical maps generated in this project will provide a valuable resource for genetic and genomic projects and studies of polyploid genome evolution. Research planned in this project parallels several other national and international projects aiming at knowledge acquisition or resource development needed for the completion of the physical maps of all 21 CS chromosomes. Synergy among these projects will greatly broaden their individual impacts. To broaden further the impact of this project, the following specific activities will be organized: nationally advertised internships for undergraduate students in each of the participating laboratories, a nationally advertised workshop at UC Davis in fingerprinting and physical mapping for graduate students and postdoctoral trainees, and a nationally advertised international internship for US graduate students and postdoctoral trainees at IEB to acquire skills in plant chromosome isolation by flow-sorting, currently unavailable in this country, and other plant molecular cytogenetic techniques. Finally, several project laboratories will host high school and junior college students supported by summer research programs in genomics and biotechnology in the home institutions.
The project database(WheatDB) will be accessible at http://wheatdb.ucdavis.edu:8080/wheatdb/index.jsp. This public database will be the initial repository of fingerprints, physical mapping information and all other deliverables generated by the project. It also provides the tools for easy access, display, and analysis of the generated data. The project data will also be integrated into GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml) and Gramene (www.gramene.org/), both curated public websites. GrainGenes provides a compilation of molecular and phenotypic information on wheat and other cereals. Gramene is an open-source, data resource for comparative genome analysis in the grasses.
A physical map of a genome is an arrayed and catalogued population of cloned DNA fragments that are ordered as to reflect the DNA sequence along a chromosome. The primary utility of a physical map is for genome sequencing employing the "ordered clone" genome sequencing approach. In this genome sequencing approach, a genome is sequenced incrementally by sequencing ordered DNA fragments making up a contiguous path of fragments along a physical map. This genome sequencing strategy is applicable for organisms with genomes that are either too large or too complex to be sequenced by other, more direct means. Many plant genomes fall into this category because the species has either originated via interspecific hybridization (polyploidy), or has a large and complex genome, or both. One of the best examples of such plant species is wheat, which originated by hybridization of three different progenitor species and has a genome that is five times the size of the human genome. In spite of a concerted international effort to sequence the wheat genome, a high quality draft of the wheat genome sequence is still unavailable. However, since the progenitor species of wheat are known, a logical strategy is to reduce the complexity of the task by sequencing the genomes of the progenitor species and use their sequences to aid the assembly of the wheat genome sequence. Building on this rationale, we undertook the construction of a physical map of the genome of Aegilops tauschii, one of the three progenitor species of wheat. Due to the large size of the Ae. tauschii genome, it was necessary to construct a physical map consisting of nearly half a million ordered DNA fragments. We accomplished this daunting task with a highly efficient molecular marker system and a computer program based on a novel algorithm that efficiently ordered DNA fragments along Ae. tauschii chromosomes. With this physical map, the Ae. tauschii genome is poised for sequencing by the "ordered-clone" sequencing strategy. In addition, we used the physical maps of the seven Ae. tauschii chromosomes as chromosome-size scaffolds into which we integrated sequences of 17,093 genes. The gene sequences made it possible to compare the Ae. tauschii genome with other cereal genomes and elucidate the evolution of the seven Ae. tauschii chromosomes (Fig. 1). The outer circle (Circle 5) of Fig. 1 shows how the seven Ae. tauschii chromosomes evolved from the 12 ancestral chromosomes by five insertions of a complete chromosome into a single break in the vicinity of the centromere of another chromosome. The active Ae. tauschii centromeres are marked with white rectangles and the centromeres of the ancestral chromosomes that went extinct are marked with black rectangles in Circle 5. The locations of current and ancient chromosome ends are indicated by the thick vertical bars in Circle 5. The physical maps also provided a framework for assessing gene distribution along the Ae. tauschii chromosomes. The physical maps showed that gene density, which is the number of genes per a unit of chromosome length, such as million base pairs (Mbp), is the lowest near the chromosome centromere and increases towards the chromosome ends (Circle 3 of Fig. 1). The physical maps also showed that meiotic recombination is the highest near the ends of Ae. tauschii chromosomes (Circle 2 of Fig. 1). These high-recombination regions are enriched for recently duplicated genes and newly evolved genes. This is indicated by the absence of a significant gene density gradient in Circle 4 of Fig. 1, based on data from which the duplicated genes were removed. This complex project provided an excellent environment for student and postdoctoral training. A total of 3 postdoctoral trainees, 4 graduate students, 35 undergraduate students, and 11 undergraduate interns received training in genomic research. To disseminate the deliverables of the project, a website with project databases was built and made publicly available (http://probes.pw.usda.gov/WheatDMarker/).
